Fig 2: Stx4 is expressed in skeletal muscles and induced in myoblast differentiation. a RT-PCR analysis of hindlimb muscles from E15.5 embryos and P1, P5, P7, P14, and P30 mice for the expression of Stx4, Cdo, MyoD, Myogenin, and 18S rRNA serves as a loading control. b Immunoblot analysis of C2C12 cells from various differentiation days (D) for the expression of Stx4, Cdo, Myogenin, MHC, and pan-Cadherin serves as a loading control. c Immunoblot analysis for Stx4 protein expression in Cdo +/+ and Cdo -/- primary myoblasts during differentiation, and pan-Cadherin serves as a loading control. d qRT-PCR analysis for Stx4 mRNA expression in Cdo +/+ and Cdo -/- primary myoblasts during differentiation

Fig 3: Overexpression or knockdown of Stx4 promotes or blocks myoblast differentiation, respectively. a Lysates of control or Stx4-overexpressing C2C12 cells were immunoblotted with antibodies against Stx4 and pan-Cadherin as a loading control. The relative signal intensity of Stx4 to pan-Cadherin was quantified and added under the blot. b Lysates of control or Stx4 expression vector transfected C2C12 cells from the differentiation day 1 (D1) and D2 were immunoblotted with antibodies to MHC, MyoD, Myogenin, Troponin T, and pan-Cadherin as a loading control. c Control or Stx4 expression vector transfected C2C12 cells were induced to differentiate for 3 days and immunostained with MHC antibody followed by DAPI staining to visualize nuclei. Size bar = 100 µm. d The quantification of myotube formation shown in panel c. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01. e Control or shStx4 expression vector transfected C2C12 cells were analyzed by immunoblotting with antibodies to Stx4 and pan-Cadherin as a loading control. The relative knockdown levels of Stx4 to pan-Cadherin are quantified and added under the blot. f Control or shStx4 expression vector transfected C2C12 cells were induced to differentiate for 2 or 3 days and then lysates were subjected to immunoblotting with antibodies to MHC, MyoD, Myogenin, Troponin T, and pan-Cadherin as a loading control. g Control pSuper or shStx4 expression vector transfected C2C12 cells were induced to differentiate for 3 days and immunostained with an antibody to MHC followed by DAPI staining to visualize nuclei. Size bar = 100 µm. h The quantification of myotube formation shown in panel g. Values represent means of triplicate determinations ± 1 SD. The experiment was repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005

Fig 4: Stx4 and Cdo interact physically in differentiating myoblasts, and the t-SNARE domain of Stx4 mediates the interaction with Cdo. a Lysates of 293T cells transfected with Stx4-myc, Cdo, or control vector were subjected to immunoprecipitation with myc and immunoblotting with Cdo or myc antibodies. b Lysates of C2C12 cells from various differentiation time courses were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to Stx4, Cdo, and pan-Cadherin as a loading control. c The schematic representation of the domain structure of Cdo. Cdo consists of five immunoglobulin, three fibronectin type III, a single transmembrane domain, and a 270-amino-acid-long intracellular region. d 293T cells were transiently cotransfected with control or Stx4-myc along with either the full length or three deletion mutants of the Cdo’s cytoplasmic region. Forty-eight hours later, cell lysates were subjected to immunoprecipitation with myc antibody followed by immunoblotting with Cdo antibody. Total lysates served as the expression controls. e The schematic representation depicts the domain structure of Stx4 and the deletion of the specific domain; ?33-153 (the Syntaxin domain deletion), ?195-262 (the t-SNARE region deletion), and ?154-194 (the linker region deletion). f 293T cells were transiently cotransfected with control or Cdo along with the full length or deletion mutants of Stx4, and the lysates were pulled down with S-agarose beads followed by immunoblotting with Cdo or GFP antibodies

Fig 5: The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 µm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 µg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, *p < 0.05, **p < 0.01